Dialysis membrane for separation on microchips

ABSTRACT

Laser-induced phase-separation polymerization of a porous acrylate polymer is used for in-situ fabrication of dialysis membranes inside glass microchannels. A shaped 355 nm laser beam is used to produce a porous polymer membrane with a thickness of about 15 μm, which bonds to the glass microchannel and forms a semi-permeable membrane. Differential permeation through a membrane formed with pentaerythritol triacrylate was observed and quantified by comparing the response of the membrane to fluorescein and fluorescently tagging 200 nm latex microspheres. Differential permeation was observed and quantified by comparing the response to rhodamine 560 and lactalbumin protein in a membrane formed with SPE-methylene bisacrylamide. The porous membranes illustrate the capability for the present technique to integrate sample cleanup into chip-based analysis systems.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of and claims benefit toSer. No. 10/443,491 filed May 22, 2003, now U.S. Pat. No. 7,264,723issued Sep. 9, 2007, entitled “DIALYSIS ON MICROCHIPS USING THIN POROUSPOLYMER MEMBRANES” which claims priority to Provisional U.S. PatentApplication Ser. No. 60/423,176 originally filed Nov. 1, 2002 and titled“DIALYSIS IN MICROCHIPS USING PHOTOPATTERNED THIN POROUS POLYMERMEMBRANES”.

STATEMENT OF GOVERNMENT INTEREST

This invention was made with Government support under governmentcontract no. DE-AC04-94AL85000 awarded by the U.S. Department of Energyto Sandia Corporation. The Government has certain rights in theinvention, including a paid-up license and the right, in limitedcircumstances, to require the owner of any patent issuing in thisinvention to license others on reasonable terms.

FIELD OF THE INVENTION

The invention is directed specifically to dialysis of chemical andbiological samples in a microfabricated device prior to analysis. Ingeneral, it relates to enabling selective control of the transport ofspecies (e.g., molecules or particles) in microfluidic channels throughthe use of a photopatterned porous membrane with controlled porestructure.

BACKGROUND OF THE INVENTION

Real-life biological, environmental or chemical samples frequentlycontain a large number of molecules of differing molecular sizes andweights. A few examples of such samples are bodily fluids such as blood,urine and saliva or the contents of a cell. The size of these particlescan range from 0.1 mm to less than 1 nm. The presence of particlesspanning such a wide range can create a number of is problems inminiaturized systems such as blockage of fluidic channels and adsorptionof unwanted molecules on system surfaces (channel fouling). Furthermore,in typical applications, it is often desirable to analyze specificclasses of molecules (e.g., proteins); eliminating other particles(e.g., cells, and cell fragments) in order to reduce the background“clutter” in the sample and thereby simplifying analysis and providinggreater sensitivity. In particular, in biomedical applications in orderto study cell proteins and signaling molecules, the cell membrane mustbe ruptured and the contents of the cell released. In practice, cellsamples are typically opened by mechanical emulsion or by exposing thecell sample to a denaturing solution. In doing so one is left with amyriad of particles and molecules that must be filtered in order to beanalyzed.

Dialysis is a means of separating molecules using a porous membrane. Theseparation is achieved according to molecular size or molecular weightof the assemblage of molecules under study: molecules smaller than themembrane pore size will pass through the membrane, while largermolecules are excluded. Dialysis, therefore, can be applied to achieveeither of two purposes: (a) to remove interfering compounds,contaminants, or salts from a biological sample; or (b) to extract thosemolecules of interest from a “dirty” sample or a crowded assemblage ofmaterials. In the former case, the molecules that do not pass throughthe membrane are of interest while in the latter case those moleculesthat do move through the membrane are of interest. The driving force fordialysis is the concentration differential between the solutions (sampleand perfusion liquid respectively) on either side of the membrane. (Forfiltration, the process is the same but the driving force is a pressuregradient.) For maximum efficiency, the membrane is made to be as thin aspossible while still providing sufficient rigidity and strength toprevent membrane rupture. Moreover, the concentration differentialacross the membrane is maintained as large as possible, and the membranepore size distribution is made as narrow as possible such that the“tails” of the distribution decline rapidly.

Microfluidic devices (specifically, those constructed using glasswet-etching, silicon micromachining, or LIGA-type processes) have inmany ways revolutionized the analytical and synthetic capabilitiesavailable for chemistry, biology, and medicine (the term “microfluidics”is herein intended to imply fluidic processes occurring in fluidchannels having cross-sectional dimensions below 1 mm and lengthsranging from millimeters to tens of centimeters). A number of analyticaltechniques have been shown to perform better in microfluidic structuresof this type, and synthesis of small structures using the minimum amountof reagents requires efficient use of materials in small channels.Microfluidic devices allow analysis using minute amounts of samples(crucial when analyzing bodily fluids or expensive drug formulations),are fast and enable development of portable systems.

When dealing with small volume samples, however, one of the majorproblems is a loss of sample due to the transfer of samples to and fromthe dialysis equipment. When sample is present in such a small volumeand not readily available the loss of sample becomes an importantconsideration.

SUMMARY OF THE INVENTION

There is a need, therefore, to develop a method and a device forperforming dialysis that does not require the transfer of samples out ofthe dialyzer and which thereby minimizes handling loss. There are manydevices currently available in the market for dialyzing small samplevolumes. However, most if not all of these devices require advancedpreparation before a sample can be dialyzed.

Moreover, a common feature of these prior art dialysis devices is theneed to transfer the sample into the dialysis device for analysis andthe need for extracting the sample from the dialysis device afterdialysis. These multi-step procedures involve an inevitable loss ofsample, are operationally complex, require prolonged analysis times, andmake integration and automation difficult and expensive.

Simultaneous miniaturization and integration of the sample pretreatmentmethods into the miniaturized analysis device not only lead tosignificant improvement in performance but also allow autonomousoperation.

An embodiment of the present invention, therefore, allows for anintegrated, miniaturized dialysis device wherein porous polymermembranes are fabricated in-situ in micro channels and used as asize-selective dialysis element to allow for species of different sizesto be distinguished, filtered, and extracted.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a cartoon of the general approach for creating amembrane by photo-initiated phase-separation polymerization.

FIG. 2 shows a photolithographic technique for beam-shaping optics toprovide the polymerized membranes.

FIG. 3A shows a schematic of intersecting microchannels and apolymerized membrane located at the intersection junction.

FIG. 3B illustrates that 200 nm (average) Ø microspheres tagged with afluorescent dye do not diffuse through the polymer membrane.

FIG. 3C illustrates fluorescein dye diffuses through the membrane.

FIG. 4A shows the migration of rhodamine 560 through the dialysismembrane after 20 seconds exposure.

FIG. 4B shows the migration of rhodamine 560 through the dialysismembrane after 160 seconds exposure.

FIG. 4C shows FITC-labeled insulin introduced on one side of thedialysis membrane at initial exposure.

FIG. 4D shows insulin on one side of the dialysis membrane 10 minutesafter initial exposure illustrating that only slightly detectablemigration of the insulin has occurred.

FIG. 4E shows FITC-labeled lactalbumin introduced on one side of thedialysis membrane at initial exposure.

FIG. 4F shows lactalbumin on one side of the dialysis membrane 12.5minutes after initial exposure illustrating that virtually no migrationof the lactalbumin has occurred.

FIG. 5 illustrates a cartoon of a 1 cm long dialysis membrane in acounter-flow channel.

FIG. 6A shows a graphical representation of the dialysis membrane in thecounter-flow channel configuration shown in FIG. 5.

FIG. 6B illustrates a dual dialysis membrane embodiment in acounter-flow channel.

FIG. 6C illustrates a dialysis membrane in a co-flow channel embodimentwherein the membrane has multiple sections with different molecularcut-off pore sizes.

FIG. 6D illustrates a tortuous dialysis membrane in a counter-flowchannel wherein the membrane is deliberately convoluted in order tolengthen the porous surface.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present embodiment consists of a means for dialyzing species in amicro-channel device that is based on the species' size. Utility isachieved by polymerizing a thin porous polymer membrane across a channelintersection within the microchannel device. A membrane of about 0.5 μmto about 20 μm in thickness can be used for this purpose. Because theshape and thickness of the membrane is controlled primarily by a UVlight beam used to initiate a polymerization reaction in a solutioncontained within a microchannel, control of the excitation light beamfocus and collimation can be used to control the membrane thickness. Thethickness of the membrane is also negatively affected by photo-initiatedradical diffusion, solvent-phase polymer diffusion, and bulk fluidmotion within the fluid microchannel. These factors can be controlled byeliminating bulk fluid flow before initiating polymerization, and by theincorporation of polymerization inhibitors to minimize radicaldiffusion.

In preparing the desired membrane, various monomers and solvents may bechosen to provide a polymerized membrane having a specific distributionof pore size. Moreover, these constituents incorporate specificmolecules into the membrane that impart a specific property to themembrane and to the membrane pore structure. Such membranes, therefore,can be adapted or “engineered” to pass molecules having a specific sizeor having a specific protein molecular weight cutoff (as measured inDalton units). Moreover the choice of monomer/solvent combinations canbe used to dictate polymer properties such as (i) pore size; (ii)mechanical strength, which can be enhanced by using high polymercross-linking density (using for example, 1% to 100% of polyfunctionalacrylates such as pentaerythritol triacrylate, polyfunctionalmethacrylate, such as 1,3 butanediol dimethacrylate, or polyfunctionalacrylamide, such as methylene bisacrylamide); (iii)hydrophobicity/hydrophilicity, which can be controlled through thechoice of monomers, e.g., ethylene glycol diacrylate, or zwitterionicmolecules, for hydrophilicity, and alkyl-acrylates for hydrophobicity;and (iv) polymer charge, which can be controlled through incorporationof charged monomers into the membrane, such as, [2-(acryloyloxy)ethyl]ammonium methyl sulfate salt (MOE) for positive charge,2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) for negative charge.

Of all of these properties, however, pore size is most common and mostimportant. By utilizing carefully chosen appropriate combinations ofmonomers and solvents such as are shown in TABLE 1, pore sizes may beadjusted from small to large in the dialysis membrane. In particular,for a given concentration of solute, solvents that are characterized as“strong” with respect to the solute monomer provide for a smalleraverage pore size upon polymerization, while solvents characterized as“weak” provide for a larger average pore size. Utilizing a monomer suchas SPE (N,N-dimethyl-N-(2 methacryloyl oxyethyl)-N-(3 sulfopropyl)ammonium betaine) and a solvent such as water, an average pose size of 1nm to 3 nm is achieved, while a monomer such as pentaerythritoltriacrylate with a solvent such as 1-propanol, the measured pore size isabout 30 nm.

TABLE 1 SOLVENT/ MONOMER/ MONOMER PORE SOLVENT CROSS-LINKER RATIO SIZE20:60:20 70:30 67:33 1000 nm Ethanol:Acetonitrile:5 mM Butylacrylate:1,3Phosphate buffer pH 6.8 Butanediol diacrylate 1-Propanol Pentaerythritol27:73 30 nm triacrylate 96:2:2 95:5 60:40 1-3 nm Water:2- SPE:N,N′-Methoxyethanol:10 mM Methylene Phosphate buffer pH 5.5 bisacrylamide

This embodiment of the invention allows for two or more liquids (onesample liquid and one or more perfusion liquids) to be brought intocontact on a microfluidic chip separated only by a thin (0.5 μm-100 μm)photopatterned porous polymer membrane; concentration gradient-drivendiffusion will cause those molecules whose size is smaller than themembrane pore size to be transported from sample through the membrane tothe perfusion liquids. Implementing this in a microfluidic chip formatallows molecules having a size range of interest to be transported toanalysis channels (e.g., chemical separation), to reaction zones(labeling, enzymatic), or to off-chip sites for mass spectrometry.

A variety of geometries may be used to implement on-chip dialysis,including co-flow and counter-flow operation, single- andmultiple-membrane configuration, straight and tortuous pathconfiguration, and both single-pass and recirculating configurations. Inparticular, FIG. 5 illustrates an example of a counter-flow geometrywherein the dialysis is 1 cm in length.

Polymer Formulation & In-situ Photopatterning of Polymer Membrane

Standard glass microchips having conventional cross-shaped channels wereobtained from Micralyne; chemicals were obtained from Aldrich and usedas received. In order to facilitate bonding between the glass surfaceswithin the channels and the polymer membrane, the glass surfaces withinthe microchannels were first exposed to a 2:2:1 (by volume) mixture ofwater, glacial acetic acid, and 3-(trimethoxysilylpropyl)acrylate for aperiod of 30 minutes, covalently linking the silane to the wall andexposing the acrylate group for polymerization.

Following surface treatment, the microchannels are filled with amonomer/solvent/photo-initiator solution comprising the followingformulation. A monomer mixture consisting of 95% (by weight) of SPE(N,N-dimethyl-N-(2 methacryloyl oxyethyl)-N-(3 sulfopropyl) ammoniumbetaine) cross-linked with 5% (by weight) N,N′-methylene bisacrylamideis prepared. The monomer mixture is subsequently incorporated into aquantity of water to yield a 40:60 monomer:solvent solution and includes0%-30% (by weight) of an organic additive to help control pore size anda small amount of a buffer solution to control the pH of the solutionmixture. In the present formulation, the organic additive was about 2%(by weight) 2-methoxyethanol, although C1-C3 alcohols or acetonitrilecould be used also) and the buffer solution was about a 2% (by weight)10 mM concentration of a phosphate buffer solution to maintain themonomer/solvent solution mixture at a pH of 5.5.

Lastly, a small quantity of a commercial grade photo-initiator is addedto the monomer/solvent solution mixture to render the solution sensitiveto UV light exposure. In the present case, the photo-initiator was2,2′-Azobis (2-methylpropionamide) dihydrochloride, purchased from WakoChemicals USA, Inc., a division of Wako Pure Chemical Industries, Ltd.,Osaka, Japan, under the trade name of V-50®. This material is added tothe monomer/solvent solution in concentrations of generally about 10mg/ml of the monomer solution and complete the polymerizable solutionformulation used to create the dialysis membrane of the presentinvention.

The other monomer/solvent solution mixture formulations are, of course,possible, including each of those listed in Table 1. Otherphoto-initiators are also possible, particularly[2,2′-Azobis-isobutyronitrile], also known as AIBN or V-40®, againpurchased from Wako Chemicals USA, Inc. However, the formulation recitedabove is preferred for practicing dialysis as described herein.

After preparing the interior surfaces of the microchannel system andfilling it with the single phase monomer/solvent solution theintersection region of the to microchannels was then exposed to afocused, collimated beam of UV laser light, shown in FIG. 2. As thisbeam of light interacts with the single phase solution aphase-separation polymerization reaction is initiated (and catalyzed bythe presence of the photo-initiator) within the cross-sectional regionof the microchannel into which the laser light is imaged. Thepolymerization reaction eventually produces the is desired porousmembrane within the microchannel as shown schematically in FIG. 1.Actual images of operational membranes are shown in FIGS. 3B and 3C aswell as FIGS. 4B-4E.

As shown in FIGS. 1 and 2, a thin (4 μm-14 μm) porous polymer membraneis fabricated in-situ in glass micro channels by projection lithography;shaping and focusing the 355 nm output of a 12 kHz, 800 ps-pulse, 160nJ-pulse, frequency-tripled Nd:YAG laser into a 1-2 μm sheet and usingthis sheet to generate photo-initiated phase separation polymerizationin the irradiated region. The thickness of the laser sheet was minimizedby spatially filtering the focused laser output beam with a 2 μm slitand imaging the resulting diffraction pattern at ˜0.5 magnification ontothe desired channel location into which the membrane is to be formed.

As noted above, a related photolithography technique is described incommonly owned U.S. patent Ser. No. 10/141,906, now U.S. Pat. No.6,952,962. However, this reference recites a contact photolithographicprocess that is inoperable in the present case. Because the imaginglight beam must propagate through roughly a millimeter of glass coveringthe embedded microchannel in which the membrane is to be formed, theincoming light is subject to degradation due to the effects ofdiffraction and dispersion. In order to overcome these problems theApplicants have adapted projection photolithography techniques forfocusing an image of the desired structure cross-section into the regionof the microchannel and thus avoiding the problems of image integrity inthe former technique as applied to the present embodiment. The processis described in greater detail in “Voltage-addressable on/offmicrovalves for high-pressure microchip separations”, (J. ChromatographyA; 979, pp. 147-154, 2002), herein incorporated by reference.

The final thickness of the membrane, however, is determined by factorsthat include more than just the optical properties of the incident laserbeam sheet.

The membrane thickness is also affected by diffusion of radical species,by solved-phase polymer diffusion, and by bulk fluid motion. Effects ofradical diffusion are reduced by retaining the natural polymerizationinhibitors present in the system (15 ppm hydroquinone monomethyl ether,solved O₂); this effectively decreases the chemical lifetime anddiffusion length of the radical products of photo-dissociation.

Laser excitation was terminated upon the onset of phase separation.Phase separation was inferred from light scattering from themembrane-fluid interface.

Following polymerization, the system was flushed thoroughly with1-propanol and water to remove residual polymer/monomer/solvent materialand then filled with aqueous solutions for testing. The nominal poresize of the present embodiment of porous polymer was established to beabout 1 nm to about 3 nm as measured with mercury porosimetry, BET, andwith SEM.

Examples of Dialysis Operation in Membranes of the Present Invention

FIGS. 3A through 3C illustrate one embodiment of the present invention.FIG. 3A shows a schematic of the channel configuration. The operation ofthe porous membrane is shown in FIG. 3C by filling the channel assemblyon one side of the polymerized membrane with an aqueous solution offluorescein (MW=0.33 kDa, Ø=1 nm); or as shown in FIG. 3B with anaqueous suspension containing 200 nm, carboxylate-modified,fluorescein-impregnated latex spheres (Molecular Probes®), while fillingthe opposite side of each of these channel assemblies with water. Bothsolutions were allowed to come to rest and the extent of speciesmigration (fluorescein or latex spheres) across the membrane observedover a period of several minutes using 488 nm light to excitefluorescence in the fluorescein. As can be seen in FIG. 3C, fluoresceinreadily diffuses across the membrane while in FIG. 3B the 200 nm latexspheres do not, suggesting that the pore size cutoff for this membraneis below 200 nm since fluorescein molecules (having a “diameter” that isabout 1 nm) pass freely through the membrane while the latex spheres areblocked. This observation is corroborated with SEM, Hg porosimetry, andBET porosimetry.

A second embodiment is shown in FIGS. 4A-F wherein the membrane, shownas element 40 diagonally separating intersecting fluid channels 41 and42, is subjected to a similar test as is illustrated in FIGS. 3B and 3C.In the present case, however, the test was modified to improve thegranularity of the attempt to determine the molecular weight cut-off ofthe SPE membrane. In this case, the microchannel system was exposed tofree dye (Rhodamine 560, MW=0.37 kDa, Ø=1 nm) and a solution containingFITC-labeled proteins with different molecular weights. In particular,the response of insulin (MW=5.7 kDa), lactalbumin (MW=14 kDa, Ø5-6 nm),bovine serum albumin (MW=66 kDa), and anti-biotin (MW=150 kDa) in theirability to diffuse through the membrane was tested. FIGS. 4A and 4B showthe rapid permeation of the Rhodamine dye through the membrane. As seenin FIG. 4B, at 20 seconds after its introduction the rhodamine dye hasalready migrated well into both arms of the fluid channels to the rightof the membrane 40. However, FIGS. 4C and 4D show that insulin (5.7 kDa)experiences only barely measurable diffusion through the membrane, andFIGS. 4E and 4F show that lactalbumin presents virtually no measurablediffusion across the membrane even after a residence time of over 12minutes. The larger species, i.e., those having MW>14 kDa, also show nodiffusion and for brevity are not shown. These preliminary results,therefore, demonstrate that control of molecular weight cutoff throughthese porous polymer membranes is achievable by precisely engineeringthe constitution of water/2-methoxyethanol solutions.

Finally, because combinations of monomers and solvents may be chosen toprovide specific pore size distributions (as noted above), those skilledin the art will realize that a dialysis device may be provided having aplurality of membranes each exhibiting a unique specific pore size whichwould allow for isolating particles in is any specific size range forany specific application. Moreover, the method described herein isapplicable to many different geometries. FIGS. 2 and 3A illustrate asimple variation of the present technique wherein the membranediagonally separates a junction made by two intersecting channels and isan example of cross-flow dialysis. FIG. 5 illustrates a counter-flowgeometry wherein the membrane divides a single channel that connects twowidely separated channel junctions by interconnecting a series ofintermediate spaced support posts. The geometry of FIG. 5 has beensuccessfully fabricated with membranes lengths of up to 1 cm.

FIGS. 6A-6D illustrate additional embodiments of the counter-flowgeometry shown in FIG. 5 wherein the membrane divides the separationchannel 60 once, in the case of FIG. 6A or twice, as in the case of FIG.6B. As before the dialysis structure is fabricated by interconnecting aseries of intermediate spaced posts 62 which bisect fluid channel 61with short segments 63 of the polymer membrane. It is also possible toconstruct a separation channel capable of selecting species having agraded series of molecular weights (sizes). As shown in FIG. 6C, whereinchannel network 60 contains groups 67 and 68 of membrane segments 63spaced out along the length of polymer membrane 69. Two groups are shownbut it is obvious that more groups could be used. The structure achievesits utility for selecting particles having more than one range ofmolecular weights when each of the segments of a particular group ofsegments is fabricated with a polymer material that has a differentaverage molecular cut-off pore size and when the groups are arranged ina logical order (ascending or descending) for its intended use. Theparticular configuration shown in FIG. 6C allows for molecular specieswith increasing molecular size to pass from the sample stream as thestream passes along the length of the membrane. While two sections areshown in FIG. 6C, in principle, any number of sections is possible.

Finally, as shown in FIG. 6D the length of the separation network ofFIG. 6A can be increased by convoluting the fluid channel. This allowsfor compact structures while still allowing for sufficient dialysislength to achieve the intended separation result.

It is, therefore, apparent that due to the flexibility of the presentprocess other geometries are possible and are limited only by theroutineer's ability to provide the necessary lithographic tools.

1. A dialysis membrane, comprising: a semipermeable polymer comprising amonomer/cross-linker/solvent solution comprising (i) a monomer selectedfrom the group consisting of alkyl-acrylates, polyfunctional acrylates,and zwitterionic molecules, (ii) a cross-linker material selected fromthe group consisting of polyfunctional acrylates, polyfunctionalmethacrylates, and polyfunctional acrylamides, (iii) a solvent medium,and (iv) a photo-initiator, wherein the semipermeable polymer isprepared by localized photo-initiated phase separation polymerization,wherein the semipermeable polymer comprises a plurality of segmentsdisposed between an inlet end and an outlet end of a microchannelnetwork fixedly attached to one or more interior surfaces of themicrochannel network and to a plurality of about equally spaced supportstructures bisecting one or more of the microchannels within themicrochannel network.
 2. The dialysis membrane of claim 1, wherein themonomer/cross-linker/solvent solution comprises a monomer/cross-linkersolution comprising a mixture of Butylacrylate and 1,3 Butanedioldiacrylate, a solvent solution comprising a mixture of Ethanol,Acetonitrile and a phosphate buffer, and a photo-initiator comprising asmall quantity of 2,2′-Azobis (2-methylpropionamide) dihydrochloride. 3.The dialysis membrane of claim 2, wherein the ratio of solvent solutionto monomer/cross-linker solution is about 67:33.
 4. The dialysismembrane of claim 3, wherein the ratio of monomer to cross-linker isabout 70:30.
 5. The dialysis membrane of claim 4, wherein the Ethanol,Acetonitrile and phosphate buffer are present in a ratio of about20:60:20.
 6. The dialysis membrane of claim 5, wherein the semipermeablepolymer comprising a pore size of about 1000 nm.
 7. The dialysismembrane of claim 1, wherein the monomer/cross-linker/solvent solutioncomprises a monomer/cross-linker solution comprising Pentaerythritoltriacrylate, a solvent solution comprising 1-Propanol, and a smallquantity of 2,2′-Azobis (2-methylpropionamide) dihydrochloride.
 8. Thedialysis membrane of claim 7, wherein the ratio of solvent solution tomonomer/cross-linker solution is about 27:73.
 9. The dialysis membraneof claim 8, wherein the semipermeable polymer comprising an average poresize of about 30 nm.
 10. The dialysis membrane of claim 1, wherein themonomer/cross-linker/solvent solution comprises a monomer/cross-linkersolution comprising a mixture of N,N-dimethyl-N-(2methacryloyloxyethyl)-N-(3 sulfopropyl) ammonium betaine and Methylenebisacrylamide, a solvent solution comprising a mixture of water,2-Methoxyethanol and a phosphate buffer, and a small quantity of2,2′-Azobis (2-methylpropionamide) dihydrochloride.
 11. The dialysismembrane of claim 10, wherein the ratio of solvent solution tomonomer/cross-linker solution is about 60:40.
 12. The dialysis membraneof claim 11, wherein the ratio of monomer to cross-linker is about 95:5.13. The dialysis membrane of claim 12, wherein the water,2-Methoxyethanol and a phosphate buffer are present in a ratio of about96:2:2.
 14. The dialysis membrane of claim 13, wherein the semipermeablepolymer comprising an average pore size of about 1 nm to about 3 nm. 15.The dialysis membrane of claim 1, wherein the semipermeable polymerfurther comprises two or more spaced groups of contiguous segments,wherein each of the spaced groups comprise a different average poresize, and wherein the widely spaced groups are arranged to place theaverage pore size in ascending or descending order.